Analytical methods for peptides: what each test can and cannot tell you

One powder, several questions

Review A vial of lyophilized peptide holds more than the target molecule: a review of impurities in peptide medicines notes that by-products of synthesis and counter-ions, the oppositely charged ions that pair with a peptide’s charged groups, may be present in the final product.⁴ The European Medicines Agency’s guideline on synthetic peptides, written for medicines, lists tests that a specification for a peptide drug substance may include, among them identification, purity, assay or content, counter-ion identity and content, residual trifluoroacetate, water content, bacterial endotoxins and microbiological purity.²

A certificate documents only the tests listed on it. The certificate of analysis guide shows how to see which were run on a batch.

Reversed-phase HPLC: purity by area

How the separation works

Liquid chromatography passes a dissolved sample through a column packed with a stationary phase, and components that the packing holds back to different degrees leave the column at different times.¹

Chemistry study Peptides are separated by size, by charge or by hydrophobicity.⁵ The last of these, the reversed-phase mode, is the routine one for quality control, traditionally on a C18 column with a gradient that contains an acid modifier such as formic acid or trifluoroacetic acid.⁶ A detector at the column outlet records the chromatogram, for example by ultraviolet absorbance at 214 nm, a wavelength at which the peptide bond itself absorbs.⁷

What “purity by area” means

The peak area percentage method takes the area of each peak as a share of the summed area of every peak in the chromatogram.¹ The Japanese Pharmacopoeia adds a condition: for an accurate result, each area has to be corrected by a factor that reflects how strongly that component responds compared with the main one.¹

Chemistry study That condition matters because amino acids do not absorb equally. In measurements at 214 nm, tryptophan absorbed about 30 times as strongly as a peptide bond, and phenylalanine, tyrosine and histidine about six times as strongly.⁷ Equal areas are therefore not always equal amounts: a by-product that lacks a tryptophan gives less signal per molecule than the full chain.

Review A review of impurities in peptide medicines sorts the peptide-related ones into those that arise during synthesis, such as chains with a missing or an extra amino acid, racemized (mirror-image) residues and leftover protecting groups, and those that form later by degradation.⁴ The guide to peptide stability and degradation covers the second group.

Reading a trace peak by peak is the subject of how to verify a peptide, and the arithmetic behind a single figure is in what a purity percentage means.

What it cannot tell you

• The International Council for Harmonisation (ICH) guideline on specifications, written for chemical drug substances, does not regard identification by a single chromatographic retention time as specific, so a purity trace alone does not establish identity.⁸
• The European guideline points to the risk of impurities that co-elute, meaning they leave the column together with another component, and asks for orthogonal or complementary procedures, such as mass spectrometry, to reduce it.²
• Chemistry study The figure depends on the system. When five cationic peptides were run on five C18 systems that differed in particle size, acid modifier and column temperature, one system outperformed the others in sensitivity and resolution, and the authors warned that the traditional method may lead to erroneous results for basic peptides.⁶

Mass spectrometry: identity

Mass spectrometry ionizes the molecules of a sample and separates the ions by their mass-to-charge ratio, written m/z.¹ The result is a mass spectrum, a plot of signal intensity against m/z, from which the molecular mass can be worked out.¹

Review Electrospray ionization produces intact ions from large and fragile molecules in solution.⁹ Its spectra have a signature: a series of peaks from the same molecule carrying different numbers of charges, each peak differing from its neighbour by one charge.⁹ A peptide of mass 1,500 that picks up two protons appears near m/z 751, and with three protons near m/z 501.

Tandem mass spectrometry breaks a selected ion apart and uses the masses of the pieces to confirm its structure, which for a peptide means its amino acid sequence.¹²

Chemistry study Coupled to chromatography, a high-resolution instrument can also measure impurities. One such method, developed at the U.S. Food and Drug Administration on three peptide drugs, was sensitive enough to detect an impurity at less than 0.1% of the main component, and it could quantify impurities even when they co-eluted.¹⁰

What it cannot tell you

Chemistry study A matching mass shows that a molecule of the expected mass is present, not how much of the sample it makes up. Nor does a mass alone separate molecules that weigh the same. A paper on peptide reference standards from the United States Pharmacopeia notes that chiral amino acids, the mirror-image D and L forms, and isobaric ones, which share a mass, may need additional techniques for full characterization.¹¹ For medicines, the European guideline recommends at least two orthogonal identification methods, meaning methods that rest on different principles.²

Amino acid analysis and net peptide content

Amino acid analysis shows how much peptide is in a weighed amount of powder. The peptide is hydrolyzed into its individual amino acids, most often with acid, and the amino acids are separated by chromatography and measured.¹ The sequence says how many of each amino acid one molecule should release, so the measured quantities give the amount of peptide. As a share of the weighed mass, that amount is the net peptide content.

Review The hydrolysis is the weak point: a review of the technique calls a properly performed hydrolysis the prerequisite of a successful analysis.¹² Under acid conditions tryptophan is destroyed, serine and threonine are partly destroyed, and asparagine and glutamine are converted to aspartic acid and glutamic acid.¹

Chemistry study For that reason the calculation commonly rests on selected, chemically stable amino acids.¹³ Hydrolysis also erases the sequence, so amino acids released from impurities are counted with those from the target. In a candidate angiotensin I reference material studied at the International Bureau of Weights and Measures, the major peptide impurities made up about 1% of the material, and leaving them uncorrected would have put a 1% error into the concentration assigned by amino acid analysis.¹³

The European guideline names elemental analysis, nitrogen determination and quantitative nuclear magnetic resonance (NMR) as other routes to content.² Why content falls below 100% is explained in peptide salts and net peptide content.

Water content: Karl Fischer titration

Karl Fischer titration measures water through a chemical reaction: water reacts quantitatively with iodine and sulphur dioxide in the presence of an alcohol and a base, so the iodine consumed gives the water present.¹ The iodine is either supplied in a reagent (the volumetric form) or generated by an electric current (the coulometric form).¹

The ICH guideline on specifications calls a water test important when a substance is hygroscopic, meaning it takes up moisture from the air, and prefers a method specific for water, such as Karl Fischer titration, over loss on drying.⁸ The result describes the sample as it was when tested.

Counter-ion analysis

Where a peptide is supplied as a salt, the counter-ion is part of what is weighed. The European guideline says the type of counter-ion should be defined and its amount controlled; acetate is usual for synthetic peptide medicines, and trifluoroacetate and chloride are also possible.²

Chemistry study A comparison of three techniques for acetate, trifluoroacetate and chloride in synthetic peptides found that ion chromatography gave the best results and that isotachophoresis had difficulty with chloride.¹⁴

Bacterial endotoxin testing

Endotoxin is lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria.³ It resists heat, and in the bloodstream it can cause fever even in very small quantities, which is why the pharmacopoeia names it as a substance to be controlled in pharmaceutical preparations.³

The classic test uses a lysate of blood cells from the horseshoe crab, Limulus polyphemus or Tachypleus tridentatus.¹ The first species gives the test its common name, LAL, for Limulus amoebocyte lysate.¹⁵ Endotoxin activates an enzyme in the lysate called factor C, which starts a chain of further enzymes that ends in a clot.³ Gel-clot techniques read the clot directly, photometric techniques follow the same reaction by turbidity or by the colour released from a synthetic substrate, and results are given in endotoxin units.¹

Recombinant reagents replace the lysate with proteins made from the crab’s gene sequences: factor C alone, or factor C with the two enzymes that follow it.³ They were developed partly to protect horseshoe crabs.³ The general information section of the Japanese Pharmacopoeia’s eighteenth edition describes them as alternative methods, for which a laboratory has to confirm accuracy, precision, sensitivity and specificity equal to or better than the lysate test.³

Review A 2020 review of comparison studies reported a high degree of correlation between recombinant factor C and lysate results.¹⁶ The lysate has a second pathway, starting from factor G, that responds to glucans, and recombinant factor C does not recognize it, which the review described as making the recombinant test more selective for endotoxin.³¹⁶

What it cannot tell you

An endotoxin result covers endotoxin only. It does not detect living organisms, and it does not detect fever-causing substances of other kinds; where those may be present, the FDA guidance points to a different test.¹⁵ The sample’s own physical and chemical properties can also interfere with the assay.¹⁵

Review In low endotoxin recovery, lysate assays fail in some cases to detect endotoxin because formulation ingredients mask it; a 2022 review describes the mechanism as not fully determined.¹⁷

Sterility testing

The sterility test asks whether living micro-organisms can be grown from a product. Samples are passed through a membrane filter or added directly to the culture media. Two media are used, one suited to anaerobic and aerobic bacteria and one to fungi and aerobic bacteria, and they are incubated for at least 14 days.¹

The pharmacopoeia states the limit of the test: a satisfactory result only indicates that no contaminating micro-organism was found in the sample examined, under the conditions of the test.¹

The test applies to material that is required to be sterile.¹ A batch whose certificate lists no sterility test has no documented sterility.

How the results fit together

Net peptide content says what share of the powder is peptide of any kind, and HPLC purity says what share of that peptide is the target. Multiplying them gives a rough estimate of the target peptide in the powder: at a net peptide content of 80% and a purity of 98%, about 78% of the powder’s mass is the target.

Chemistry study The mass balance approach runs the same logic from the other side: estimate the mass fraction of everything that is not the main peptide, and assign the remainder to the peptide.¹³ The two routes can be checked against each other. In a 2026 study that assigned a reference value to the peptide sublancin, subtracting the measured water, trifluoroacetate and other impurities left 78.60%, and an independent measurement based on amino acid analysis gave 77.58%.¹⁸

Human evidence

None of these methods measures what a compound does in a living system. Purity, identity, content, endotoxin and sterility results describe a material; they are not evidence that a compound has an effect, or is safe, in people, and this page cites no human study. The guide to levels of evidence in peptide research explains how studies in cells, animals and people are weighed.