Reading an Amino Acid Profile: Units, Ratios and Flags

To interpret amino acid analysis results, settle four things before any biology: the unit, the ratio basis, the flags, and what "as received" means. This page walks through our example report to show each one. The report is watermarked EXAMPLE. Its values are illustrative, chosen to be internally consistent; they are not measurements of any sample.

The example sample

The example is spent medium from a fed-batch culture, sampled at 48 h. The results table lists 19 amino acids plus ammonia, each in µM and mg/L, with a Flag column. A second page carries the QC summary for the run and a section for the chromatogram. The AA-1 profile on our amino acid analysis service reports the same analytes in the same two units.

µM and mg/L: counting versus weighing

A µM value counts molecules; a mg/L value weighs them. The conversion is:

mg/L = µM × molecular weight (g/mol) ÷ 1000

Glutamine has a molecular weight of 146.14 g/mol ¹. The example's 1842 µM glutamine therefore becomes 1842 × 146.14 ÷ 1000 = 269.2 mg/L.

The report uses free amino acid molecular weights, because free amino acids are what we measure. Protein composition work uses weights corrected for the water lost in each peptide bond ^2,3. Mixing the two bases gives inconsistent numbers, so check which one any comparison value uses.

The unit changes how a table reads. Ammonia is the largest number in the example at 2260 µM, yet only 38.5 mg/L, because its molecular weight is 17.03 ⁴. Glycine (455 µM) outnumbers arginine (388 µM) by molecules. By mass the order flips: arginine is 67.6 mg/L, glycine 34.2 mg/L.

Use µM for stoichiometry: how much of one compound became another, or where nitrogen went. Use mg/L when you compare against a recipe weighed in grams. Amino acid results are routinely expressed on molar, mole-percent or weight bases, depending on the application ³. Winemakers also meet mg N/L, the unit for yeast assimilable nitrogen ⁵. That basis counts nitrogen atoms, so it is not interchangeable with mg/L of an amino acid.

Mol% and why ratios matter

Mol% expresses each amino acid as a share of the molar total. The pharmacopeial chapter on amino acid analysis defines it for proteins as residues of one amino acid per 100 residues ². For a free amino acid profile, it is each amino acid's share of all amino acids measured. In the example, the 19 amino acids sum to 7603 µM. Glutamine is 24.2 mol% and alanine 14.7 mol%. We leave ammonia out of that total because it is not an amino acid.

Ratios are immune to anything that scales every analyte equally. A dilution or volume error shifts every µM value, but not the mol%. That makes ratios the better basis for comparing samples of different strength, such as two lots of a hydrolysate. The same chapter notes that mole percent is useful when a protein's molecular weight is unknown ².

In cell culture, ratios are also process variables. One CHO study changed the asparagine-to-glutamine ratio in feed media and reported differences in growth, antibody production and metabolic waste ⁶.

What a "< LOQ" flag means

The quantitation limit is the lowest amount of analyte that can be determined with suitable precision and accuracy ⁷. Below it, a signal may still be detectable, but it cannot be given a reliable value ⁷. That is why we flag such results instead of reporting them as numbers.

Treat a flagged result as "below the quantitation limit", not as zero and not as the limit itself. If you substitute a value in later calculations, record that you did. In the example, nothing is flagged: tryptophan is the smallest result at 24 µM and is reported as a number. The quality control page explains how flagging fits with our other checks.

"As received": dilution correction

Every concentration on the report refers to the sample as received. We dilute in-house so that each analyte falls within the calibrated range. The example was diluted 1:10 before derivatization, so the instrument saw about 184 µM glutamine; the report multiplies back to 1842 µM. ICH Q2(R2) draws the same distinction between the working range presented to the instrument and the reportable range after sample preparation ⁷.

Dilution has one consequence worth knowing. A 1:10 dilution raises the effective quantitation limit in your original sample tenfold. A minor analyte in a concentrated, heavily diluted sample can therefore be flagged even though it is present. The sample preparation page covers how dilution is chosen.

What the chromatogram is for

The chromatogram is the evidence behind the table. Peaks are identified by retention time against standards, and integration is checked before quantitation ³. The trace shows what the numbers cannot: a shoulder on a peak, an unexpected peak, a noisy baseline. Analytical guidance calls this selectivity, the extent to which other substances interfere with an analyte ⁷.

Two peaks that do not come from your sample are expected. Excess AQC reagent hydrolyzes to 6-aminoquinoline (AMQ), a non-interfering by-product ^8,9. An ammonia peak is always present too, and its size varies with ammonia in the water and air ¹⁰. In the example report the chromatogram section is a placeholder; real reports include the trace for each sample. For the chemistry, see AQC derivatization.

Reading a time course: consumption versus secretion

One sample is a snapshot; a time course shows direction. A falling concentration means net consumption, a rising one net production. Specific rates are calculated from the change in concentration over time relative to viable cell density ¹¹.

Glutamine needs care, because it also breaks down without cells. It forms pyroglutamate and ammonia at a rate that depends on pH, temperature and the anions present; phosphate and bicarbonate speed it up ^12,13. Some glutamine loss and ammonia gain can therefore be chemical. One fed-batch study judged that decomposition negligibly slow next to cellular uptake ⁶. Whether that holds for your process depends on its conditions.

Ammonia typically rises in mammalian cell culture, mainly from consumption of glutamine and other amino acids ¹⁴. Cells deaminate asparagine as well as glutamine ⁶. Alanine serves as a nitrogen sink: alanine transaminase converts glutamate and pyruvate to alanine and α-ketoglutarate ¹⁴. Alanine can switch from production to consumption later in culture. In one CHO batch study it did so from the start of the stationary phase. The authors note this switch has been linked to asparagine and aspartate running out ¹¹.

Rising values need context too. In one fed-batch study, glutamine fell during exponential growth, held steady, then rose from day 8 while feed kept supplying it ⁶.

Read the example against that literature. At 48 h, ammonia is 2260 µM, alanine 1120 µM and glutamine still 1842 µM. That pattern fits active glutamine metabolism with alanine taking up nitrogen. But one time point cannot show direction; you need the fresh medium, the feed composition and earlier samples. Our note on glutamine, glutamate and ammonia in fed-batch culture goes further. The analyte reference covers stability analyte by analyte.

References

1. L-Glutamine — National Institute of Standards and Technology, NIST Chemistry WebBook.
2. 〈1052〉 Biotechnology-Derived Articles—Amino Acid Analysis — United States Pharmacopeia, harmonized text, official 2018.
3. Quantitation of Amino Acids — Waters Corporation, Comprehensive Guide to Hydrolysis and Analysis of Amino Acids (primer).
4. Ammonia — National Institute of Standards and Technology, NIST Chemistry WebBook.
5. Yeast assimilable nitrogen — S.M. Weeks and P.A. Henschke, The Australian Wine Research Institute (from Australian & New Zealand Wine Industry Journal), 1999.
6. Responses of CHO-DHFR cells to ratio of asparagine to glutamine in feed media: cell growth, antibody production, metabolic waste, glutamate, and energy metabolism — L.-X. Zhang et al., Bioresources and Bioprocessing, 2016.
7. Validation of Analytical Procedures Q2(R2) — International Council for Harmonisation, 2023.
8. UPLC Amino Acid Analysis Solution System Guide, Rev. B — Waters Corporation, 2007.
9. Fast and Sensitive Quantification of AccQ-Tag Derivatized Amino Acids and Biogenic Amines by UHPLC-UV Analysis from Complex Biological Samples — A. Guba et al., Metabolites, 2022.
10. What is the source of the NH3 peak in the AccQ•Tag derivatization reaction? — Waters Corporation, Knowledge Base.
11. mAb production kinetics in CHO batch culture: exploring extracellular and intracellular dynamics — A. Avilan Garzon et al., Frontiers in Bioengineering and Biotechnology, 2025.
12. L-Glutamine in Cell Culture — MilliporeSigma, technical article.
13. Amino acids in the cultivation of mammalian cells — A. Salazar, M. Keusgen and J. von Hagen, Amino Acids, 2016.
14. Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles — B.F. Synoground et al., Biotechnology Journal, 2021 (author manuscript via NSF Public Access Repository).

OpenChemWorks Laboratory · Reviewed by the laboratory director, PhD chemist · Published September 23, 2026