AQC is a one-step reagent that turns each amino acid, proline included, into a stable urea derivative that a C18 column can separate and a UV detector can read at 260 nm. The chemistry is forgiving, but it needs the right pH and enough reagent for every amine in the vial.
AQC derivatization attaches the same chemical tag to every amino acid in a sample in one step, so that reversed-phase HPLC on a C18 column can separate them and UV detection at 260 nm can measure them. It is the chemical core of every free amino acid analysis we run, and several common sources of error trace back to it.
What AQC is
AQC is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, a reagent described in 1993 for HPLC amino acid analysis 1. The original paper reported that it reacts with amino acids in a rapid, one-step procedure to form stable unsymmetric urea derivatives 1. Waters sells the reagent in its AccQ•Tag and AccQ•Fluor kits, and its documentation describes the working procedure in detail 3,4.
Every derivative carries the same aminoquinolyl-carbonyl tag. In mass spectrometry, that shared tag produces a common fragment ion at m/z 171 9. For chromatography, the tag makes the amino acids hydrophobic enough to retain and separate on a C18 column 9.
The reaction and its by-products
AQC reacts with both primary and secondary amino groups 2,3. The reagent is added in excess, and whatever does not find an amine reacts with water instead 2,3. That hydrolysis produces 6-aminoquinoline (AMQ), N-hydroxysuccinimide (NHS) and carbon dioxide 2. AMQ then reacts with leftover reagent to form a stable bis-aminoquinoline urea 2,3.
None of this needs cleaning up before injection. The original paper found AMQ to be the only major fluorescent by-product, with no significant interference, and injected the reaction mixture directly 1. Waters states that the side products do not interfere with separation, identification or quantitation 3. It also notes that excess reagent hydrolyzes on its own, so no extraction step is needed 5.
Why borate buffer and pH matter
An amino group has to be unprotonated to react. Waters' kit manual puts it simply: when pH is too low, "only unprotonated amines will react" 10.
The original study reported excellent derivative yields from pH 8.2 to 10.0 1. Waters gives an optimum of 8.2 to 10.1 and supplies a borate buffer to hold the reaction in that window 3.
Acidic samples are the usual threat. Waters' primer notes that a sample in 0.1 N HCl can go into the reaction in small volumes without adjustment, while stronger acid should be neutralized first 3. It also gives a visual check: if the mixture turns bright yellow when reagent is added, the pH is too low 3.
The heating step
Published AQC protocols heat the reaction at 55 °C for 10 minutes after the reagent is added 2,9,10. According to the reagent manual, the reason is tyrosine. Heating converts a minor tyrosine side product into the main mono-derivatized form 4. At room temperature the same conversion is slower, with a half-life of about one hour 4. Skipping the heat would leave the tyrosine result dependent on how long the vial had been sitting.
How stable the derivatives are
The ureas AQC forms are stable 1. Waters reports that derivatized samples stay stable for several days 5. Its reagent manual adds that tightly sealed vials, protected from evaporation, can be kept at room temperature for up to one week 4. Stability is what makes repeat injection of the same derivatized sample possible 3.
Detection: UV at 260 nm, fluorescence in the literature
We detect AQC derivatives by UV absorbance at 260 nm. Waters uses that wavelength in its UV methods for hydrolysates and cell culture media 5,6. So do a peer-reviewed UHPLC-UV method for complex biological samples 2 and a Thermo Fisher method for cell culture medium 7.
The same derivatives fluoresce, and fluorescence detection is common in the literature. The original paper used excitation at 250 nm and emission at 395 nm 1. The Thermo Fisher note ran UV at 260 nm alongside fluorescence at 266 nm excitation and 473 nm emission 7. Mass spectrometric detection of AQC derivatives is also published 9.
Why proline is covered
Proline is a secondary amino acid 8. That matters because some derivatization reagents miss it. o-Phthalaldehyde (OPA), another pre-column reagent, does not react with secondary amino acids such as proline 8. OPA methods therefore add a second reagent, FMOC, to pick them up 8.
AQC reacts with primary and secondary amines in the same step 2,5. So proline is derivatized in the same vial and measured in the same run as the other 18 amino acids in our AA-1 panel. Hydroxyproline, in the AA-2 extended panel, is measured the same way. For how AQC compares with other chemistries, see choosing an amino acid method.
What this means for your results
Reagent excess
Every amine in the vial competes for reagent, not only the amino acids you want reported. The original paper saw maximal yields at a molar excess of about three or more 1. Waters recommends a 4–6× molar excess over total amines 3.
Not every amino acid reacts to a shortfall equally. Waters notes that alanine is significantly affected by insufficient excess, while phenylalanine is more robust 3. A reagent-starved sample can therefore distort the profile, not just shrink it.
This is why we dilute samples into the working range in-house (we expect 5 µM to 5 mM per analyte). It is also why protein is removed before derivatization, as explained in sample preparation.
pH control
Samples that carry acid can drag the reaction below its working pH 3,10. Samples held in hydrochloric acid are the case Waters addresses directly 3; acid-precipitated extracts raise the same issue. A low-pH reaction under-derivatizes, and the loss is not uniform across amino acids 3. Getting sample pH into range before the reagent goes in is part of preparation, not an afterthought.
Salts, detergents and matrix effects
The reaction itself is tolerant. The original study found no discernible drop in yield from common buffer salts and detergents, provided the sample was well buffered 1.
The matrix can still affect the final number. A UHPLC-UV study of serum and tears found a considerable matrix effect and recommended calibrating in the matching matrix 2. We address this with the controls described on our quality control page: an internal standard in every sample, reagent blanks, a QC standard of known concentration with every run, detector sensitivity checks, and spiked amino acids where the matrix calls for a recovery check. The first time we see a new matrix type, a spike-recovery qualification (PREP-2) is available. Results below the quantitation limit are flagged, not reported as numbers.
References
- Synthesis of a Fluorescent Derivatizing Reagent, 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate, and Its Application for the Analysis of Hydrolysate Amino Acids via High-Performance Liquid Chromatography — Cohen SA, Michaud DP, Analytical Biochemistry 211(2):279–287, 1993.
- Fast and Sensitive Quantification of AccQ-Tag Derivatized Amino Acids and Biogenic Amines by UHPLC-UV Analysis from Complex Biological Samples — Guba A, Bába O, Tőzsér J, Csősz É, Kalló G, Metabolites, 2022.
- Derivatization of Amino Acids Using Waters AccQ•Tag Chemistry — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer), n.d.
- AccQ•Fluor Reagent Kit Care and Use Manual (WAT0052881) — Waters Corporation, care and use manual, n.d.
- UPLC Amino Acid Analysis Solution — Wheat TE, Grumbach ES, Mazzeo JR, Waters application note, 2008.
- Monitoring Cell Culture Media for Amino Acids Using an ACQUITY UPLC H-Class PLUS System — Maziarz M, Waters application note, 2018 (revised 2022).
- Amino acid analysis of mammalian cell culture medium by liquid chromatography with UV and fluorescence detection and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate — Thermo Fisher Scientific, Application Note 73057, 2019.
- Analysis of Amino Acids by On-line Pre-column Derivatization with OPA and FMOC using RHPLC System — JASCO, application page, n.d.
- A novel screening method for free non-standard amino acids in human plasma samples using AccQ·Tag reagents and LC-MS/MS — Ng DHJ et al., Analytical Methods (Royal Society of Chemistry), 2023.
- AccQ•Tag Ultra Derivatization Automation Kit (Hamilton) Care and Use Manual (720006661EN) — Waters Corporation, care and use manual, 2020.
OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Published September 23, 2026