Before we derivatize, we remove protein from your sample, bring it into the working range and add an internal standard. Each deproteinization route has trade-offs in pH, dilution and losses, and the right one depends on the matrix.
Protein has to come out of a sample before we can measure its free amino acids, because the derivatizing reagent reacts with amino groups wherever it finds them. This page explains what happens to a sample between arrival and injection, and why each step is there. We do this preparation in-house; nothing here is a set of instructions for you.
Why protein has to come out first
Our method tags amino acids with AQC, which reacts with primary and secondary amines 1,3. The reagent must stay in molar excess over all the amines in the reaction; Waters specifies a 4–6× excess for complete derivatization 2. Proteins and peptides carry amino groups of their own. Left in the sample, they spend reagent on material that is not free amino acid. The chemistry is covered in detail on our AQC derivatization page.
Protein interference is a general problem when small molecules are measured in biological samples 5. Published free amino acid methods therefore remove it first. A Waters method for physiological amino acids states that plasma requires deproteinization before analysis 4. A UHPLC-UV method for serum and tears passed samples through 3 kDa cut-off spin columns before derivatizing 1.
Particulates and lipids are a separate issue. Waters recommends centrifugation when a sample carries large amounts of either 2.
The three main routes
| Approach | How it removes protein | Main trade-off |
|---|---|---|
| Ultrafiltration (molecular-weight cut-off) | A membrane holds back large molecules | Possible losses to the filter; slower 2,5 |
| Acid precipitation (SSA, PCA, TCA) | Strong acid precipitates protein | Acidic extract must be neutralized; added volume 3,5 |
| Organic solvent (e.g., cold methanol) | Solvent precipitates protein | Large dilution 7 |
Ultrafiltration
Spin filters with a molecular-weight cut-off let small molecules through and hold proteins back. In the serum and tears study, 3 kDa filters produced no significant difference in peak areas between filtered and unfiltered samples 1. Ultrafiltration adds no acid and no reagent, so it leaves sample pH and volume largely unchanged.
The caveats are practical. Waters notes that recoveries can depend on the filter material 2. A vendor deproteinization protocol describes 10 kDa filtration as effective but more time-consuming than acid precipitation 5.
Acid precipitation
Adding a strong acid precipitates protein, which is then spun down. Three acids appear in the sources we reviewed:
- Sulfosalicylic acid (SSA). Waters' physiological amino acid method mixes plasma 1:1 with 10% SSA that already contains the internal standard, then centrifuges 4.
- Perchloric acid (PCA). A vendor protocol brings PCA to 1 M, then neutralizes the supernatant with potassium hydroxide, which also precipitates excess PCA 5.
- Trichloroacetic acid (TCA). The same protocol describes TCA as harsher than PCA 5.
Acid precipitation has three costs. The first is pH. AQC needs pH 8.2–10 3, and when pH is too low only unprotonated amines react 8. Waters advises neutralizing samples that carry more than dilute acid before derivatization 2.
The second is dilution. Each addition, acid and then base, dilutes the sample, and the result has to be corrected for it. The PCA protocol gives the arithmetic explicitly 5.
The third is incomplete removal. In a comparison of precipitants applied to a protein hydrolysate, 88%, 79% and 69% of the nitrogen stayed in solution after SSA, TCA and tungstic acid 6. The peptides left in the supernatants averaged 330–380 daltons 6. Some intact proteins needed high acid concentrations: β-lactoglobulin required 10% TCA or 20% SSA to precipitate fully 6.
Organic solvent precipitation
Cold organic solvent precipitates protein without adding acid. One LC-MS/MS method for plasma amino acids precipitated protein with 80% cold methanol at a 1:10 ratio before AQC derivatization 7. The trade-off is a tenfold dilution at the start. That suits concentrated samples and costs sensitivity in dilute ones.
Getting into the working range
After protein removal, we dilute the extract so every analyte lands inside the calibrated range. We expect samples at 5 µM to 5 mM per analyte and handle the dilution in-house.
Dilution does two jobs. It keeps the reagent in excess 1,2. It also limits how much acid or salt reaches the borate buffer; the AQC reaction tolerates common salts when the sample is well buffered 3.
Published methods scale dilution to the matrix. Thermo Fisher diluted cell culture medium 100-fold before derivatization 11. A Waters beverage study recommends preparing samples undiluted and at 1:10, 1:20 and 1:100 to keep every amino acid within the calibration range 12. Running more than one dilution is how a wide concentration spread gets covered.
The internal standard goes in first
We add an internal standard to every sample. Where it goes in matters, because it only tracks losses that happen after it is added.
Waters' plasma method puts norvaline into the SSA precipitant itself, so the internal standard passes through precipitation and every later step 4. The AOAC total amino acid method for infant formula adds norvaline at the hydrolysis step for the same reason 10. Waters describes an internal standard as the best way to compensate for variability introduced by sample hydrolysis and the analysis itself 9.
The internal standard works alongside our other controls: 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. Details are on our quality control page.
Matrix by matrix
Cell culture media and fermentation broths
Spent medium and broth usually need light preparation. Thermo Fisher notes that free amino acids in fermentation broth require only filtration and, in some cases, protein precipitation 11. Glutamine is the analyte to watch. It decomposes over time at physiological pH, separately from cell uptake 11. See glutamine, glutamate and ammonia in fed-batch and our cell culture media page.
Protein hydrolysates
Hydrolysates contain free amino acids, small peptides and large peptides, in proportions that vary with the protein, enzyme and process 13. Deproteinization removes the large material, but small peptides can stay in solution 6. Our result reports free amino acids; amino acids still bound in peptides are not part of it. The distinction is explained in free vs total amino acids and in our note on free amino acids in hydrolysates.
Juices, wine and beer
Clear beverages are among the simplest matrices. In the Waters beverage study, preparation before derivatization was dilution alone 12. See wine and brewing.
Solids, powders and new matrices
Solids and powders must be brought into solution before any of the steps above; that extraction is our PREP-1 service. The first time we see a new matrix type, PREP-2 runs a spike-recovery check on it. Both are listed on our pricing page.
References
- 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.
- 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.
- Analysis of Physiological Amino Acids With The MassTrak Amino Acid Analysis Solution — Hong P, Wheat TE, Diehl DM, Waters application note, 2009.
- Deproteinization (PCA deproteinization protocol) — Abcam, technical protocol, n.d.
- Comparison of the abilities of trichloroacetic, picric, sulfosalicylic, and tungstic acids to precipitate protein hydrolysates and proteins — Greenberg NA, Sipe W, Journal of Food Science 44:735–737, 1979 (abstract via EurekaMag).
- 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.
- Amino Acid Internal Standard, Norvaline (186009301) — Waters Corporation, product page, n.d.
- Total Amino Acids by UHPLC-UV in Infant Formulas and Adult Nutritionals — Jaudzems G, Guthrie J, Lahrichi S, Fuerer C, Journal of AOAC International 102(4), 2019 (pre-print).
- 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.
- Determination of Free Amino Acids Content in Alcoholic and Non-Alcoholic Beverages Using the AccQ•Tag Ultra Derivatization Kit — Barknowitz G, Henry C, Gould D, Foddy H, Waters application note, 2022.
- Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance — Hou Y, Wu Z, Dai Z, Wang G, Wu G, Journal of Animal Science and Biotechnology, 2017.
OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Published September 23, 2026