Underivatized Amino Acid Analysis by LC-MS/MS: Methods and Column Strategies

Amino acid analysis is a cornerstone of clinical diagnostics, food science and metabolomics — but amino acids are notoriously awkward analytes. For decades the standard approach relied on derivatization: chemically tagging each amino acid before analysis to make it retain and detect better. Modern LC-MS/MS increasingly does away with that step, analysing amino acids underivatized (in their native form). This guide explains why, what the analytical challenges are, and which chromatographic strategies work — with links to recent, peer-reviewed methods.

Amino acids are a classic example of polar analytes, so the fundamentals in our HILIC column guide for polar metabolites apply directly here.

Why derivatization was the traditional route

Underivatized amino acids present three intrinsic problems for chromatography:

• Poor UV/fluorescence detection. Most amino acids lack a chromophore or fluorophore, so they respond weakly to conventional UV and fluorescence detectors.
• Weak retention on reversed-phase. Their high polarity means they barely retain on C18, eluting unresolved near the void volume.
• Isobaric and isomeric overlap. Leucine, isoleucine and allo-isoleucine share the same mass and must be chromatographically resolved [1].

The historical gold standard — ion-exchange chromatography with post-column ninhydrin derivatization — solved detection but at a cost: long run times and limited throughput. Pre-column derivatization (with reagents such as AQC or dansyl chloride) sped things up but added sample-prep steps, reagent costs and potential side reactions. A validated 2022 method using pre-column Fmoc derivatization illustrates what that route still requires in practice — and how much of the method development goes into the mobile-phase additives [2].

Why go underivatized?

Coupling LC to tandem mass spectrometry (LC-MS/MS) changes the equation. A mass spectrometer detects amino acids directly by their mass transitions, so the chromophore problem disappears — and with it, the main reason for derivatizing. The benefits are substantial:

• Faster, simpler workflows — minimal sample prep, often just protein precipitation, and no derivatization reaction to control.
• Fewer error sources — no incomplete or variable derivatization, no unstable derivatives.
• High sensitivity and selectivity from MS/MS multiple-reaction monitoring (MRM/SRM).
• Small sample volumes — validated methods have been reported using only about 20 µL of sample [3].

The remaining challenge is chromatographic: how do you retain and separate these highly polar, native molecules well enough for reliable quantification? Three strategies dominate.

Strategy 1: HILIC

Hydrophilic interaction chromatography is the most common route. Its polar stationary phase retains amino acids that reversed-phase cannot hold, and its high-organic mobile phase boosts electrospray sensitivity. A widely cited clinical method by Prinsen and colleagues separated 36 underivatized amino acids in plasma on a Waters ACQUITY BEH Amide column in an 18-minute run, correctly identifying patients with inborn errors of metabolism [4].

More recently, a fully validated HILIC-based LC-MS/MS method for quantifying natural amino acids in human plasma reported better retention and peak symmetry for all analytes and was applied to ovarian-tumour patient samples [5].

HILIC’s known weaknesses — long equilibration, sensitivity to sample solvent, and sometimes incomplete separation of isobaric pairs — make method conditions critical. Retention here is not driven by partitioning alone: hydrogen bonding and electrostatic interaction with the surface both contribute, which is why buffer concentration and pH change selectivity as much as the gradient does [6]. A phase developed specifically for this application is the Agilent InfinityLab Poroshell 120 HILIC-Z, widely used for underivatized amino acids in plasma and plant matrices [7]. Our mobile phase basics guide and the HILIC method development guide cover the buffer and solvent choices that matter most here.

Strategy 2: Mixed-mode chromatography

Mixed-mode columns combine reversed-phase with ion-exchange (or other polar) interactions on a single phase, giving strong, tunable retention for zwitterionic amino acids without ion-pairing reagents. This approach has become popular in clinical laboratories precisely because it resolves the difficult isobaric species.

A clinically validated method quantified 38 underivatized amino acids and related compounds in both plasma and urine in under 15 minutes, resolving leucine, isoleucine and allo-isoleucine — a key requirement for diagnosing metabolic disorders [1]. For the specific challenge of branched-chain amino acids in maple syrup urine disease, a rapid mixed-mode LC-MS/MS assay using an Imtakt Intrada Amino Acid column achieved the needed isomer separation with only 20 µL of sample [3]. The Intrada Amino Acid phase is purpose-built for LC-MS analysis of intact (underivatized) amino acids and is specifically designed to resolve leucine and isoleucine [8].

Strategy 3: Ion-pairing reversed-phase

Adding an ion-pairing reagent to a reversed-phase method lets C18 columns retain amino acids by pairing with their charged groups. It works and can give excellent separation, but it carries well-known downsides: ion-pairing reagents cause ionisation suppression, shift retention times and effectively dedicate the LC-MS system to one method because they are so hard to wash out. The same mechanism is documented for trifluoroacetic acid, where a switch to a weaker acid increased protein detectability by 35–160 times [9]. For that reason many labs now prefer HILIC or mixed-mode. A 2023 ion-pairing LC-MS/MS method for sulfur-containing amino acids and related compounds shows what the approach can still deliver [10].

The isobaric separation problem

Whichever strategy you choose, one requirement recurs in almost every serious amino acid method: chromatographically separating leucine, isoleucine and allo-isoleucine. These share an identical mass, so the mass spectrometer alone cannot distinguish them — the chromatography must. Allo-isoleucine in particular is a diagnostic marker for maple syrup urine disease, so its clean resolution is clinically essential [1][3].

This is also the limit of high resolution: a mass analyser separates species by mass, and true isomers have none to separate. No amount of resolving power replaces the separation — a point developed further in our article on how an Orbitrap works. When you evaluate any underivatized amino acid method, check first how well it handles this trio; it is the acid test of the separation.

Choosing an approach

• HILIC — great MS sensitivity and broad coverage; watch equilibration and isobaric separation.
• Mixed-mode — strong, tunable retention and excellent isomer resolution; a favourite in clinical labs.
• Ion-pairing RP — powerful separation but dedicates the system and suppresses ionisation; used more selectively now.

The right choice depends on your matrix, your target panel and whether isobaric resolution is critical — the same column-selection logic as in our guide on how to choose the right HPLC column.

Key takeaways

• Underivatized amino acid analysis by LC-MS/MS removes the slow, error-prone derivatization step.
• MS detection solves the missing-chromophore problem; the remaining challenge is retaining and separating these very polar molecules.
• HILIC and mixed-mode are the leading strategies; ion-pairing RP works but has drawbacks.
• Resolving the isobaric leucine/isoleucine/allo-isoleucine trio is the benchmark of a good method.
• Recent validated methods confirm underivatized LC-MS/MS as a fast, sensitive, clinically robust standard.

References

1. Quantification of 38 underivatized amino acids and related compounds in plasma and urine by mixed-mode LC-MS/MS in under 15 minutes, resolving leucine, isoleucine and allo-isoleucine. Molecular Genetics and Metabolism. Open-access article (PMC9142622)
2. Kahsay BN et al. Development and validation of an RP-HPLC/DAD method for the simultaneous analysis of 18 free amino acids in topical formulations. Chromatographia. 2022;85 — example of a validated derivatization-based method and the role of mobile-phase additives. doi:10.1007/s10337-022-04160-0
3. Rapid mixed-mode LC-MS/MS assay for branched-chain amino acids in maple syrup urine disease, using 20 µL of sample. Open-access article (PMC9120951)
4. Prinsen HCMT et al. Rapid quantification of underivatized amino acids in plasma by HILIC-MS/MS. Journal of Inherited Metabolic Disease. 2016. Open-access article (PMC4987396)
5. Validated HILIC-based LC-MS/MS method for natural amino acids in human plasma. Journal of Pharmaceutical and Biomedical Analysis. 2025. Article page
6. Guo Y, Muscatiello D. An update on the progress in fundamental understanding of hydrophilic interaction liquid chromatography. Journal of Chromatography A. 2026;1765:466520. doi:10.1016/j.chroma.2025.466520
7. InfinityLab Poroshell 120 HILIC-Z columns — product information. Agilent. agilent.com
8. Intrada Amino Acid column — product information for LC-MS analysis of intact amino acids. Imtakt. imtakt.com
9. Huber CG, Premstaller A. Evaluation of volatile eluents and electrolytes for HPLC–ESI-MS of proteins. Journal of Chromatography A. 1999;849 — ion-pairing acids and their effect on electrospray response. doi:10.1016/S0021-9673(99)00532-4
10. Ion-pairing LC-MS/MS method for sulfur-containing amino acids and related compounds. Journal of Chromatography B. 2023. Article page