Peptide water content: Karl Fischer testing and why water changes net peptide content

Peptide water content: how much water a freeze-dried powder holds

A freeze-dried peptide is never completely dry. In a 2023 paper led by USP scientists on peptide reference standards, six lyophilised candidate lots held between 1.11% and 2.79% water by weight. The lots were made to be reference materials: dissolved in water, filled into glass vials, freeze-dried and stoppered. Powder made, sealed or stored another way can hold a different amount, so a lab measures water rather than assuming it.

The amounts are small in absolute terms. A cake of 10 mg of powder at 2.5% water holds 0.25 mg, or 250 µg, of water. That scale decides which kind of Karl Fischer (KF) titration fits a single vial.

Why peptide powder takes up water

Peptides are often hygroscopic. Bachem’s handling notes advise warming a peptide to room temperature in a desiccator before opening it, then weighing it out and resealing it without delay. Bachem adds that adsorbed water may lower stability and reduce the overall peptide content. Metrohm’s KF monograph makes the same point from the lab side: hygroscopic samples carry a large amount of water on their surfaces, and moisture from the air is a constant source of contamination.

A water result therefore describes the powder at the moment the lab opened the vial. PharmLabs’ shipping instructions ask for vials to stay sealed in their original containers, because a compromised seal in transit changes moisture content.

Regulators treat residual moisture as a quality attribute. The FDA’s inspection guide on lyophilised parenterals calls the moisture in vials an obvious concern, and asks for stability data from the batches with higher moisture. The EMA guideline on synthetic peptides lists water content in the specification of a peptide active substance, next to counter-ion content, residual TFA and residual solvents.

How water changes net peptide content

Water adds weight and no peptide. Bachem defines net peptide content as the fraction of peptidic material relative to counter-ions and residual water. Our guide on net peptide content covers the full calculation. This table shows which COA numbers water touches.

Result on a COA	Does water change it?	Why
HPLC purity (% area)	No	Purity counts UV-absorbing peaks; water gives none
Net peptide content (% w/w)	Yes	Water is part of the powder’s weight
mg per vial, whole vial measured against a reference standard	Not directly	The peptide in the vial stays the same as the cake gains or loses water
mg from powder weight × purity	Yes	The weighed water is counted as if it were peptide
Content of the reference standard	Yes	A weighed standard includes its own water

The last row matters most. The USP authors measured water at the time each HPLC preparation was made, because humidity differs between labs. They then corrected purity for water as purity × (100 − water %) ÷ 100. The arithmetic shows why. If a lab weighs a standard that holds 3% water and does not correct for it, every sample result calibrated against that standard reads about 3% high. The EMA guideline expresses the limits of LC assays on a counter-ion-free, anhydrous basis.

Few reports say which basis they use. In HK Analytical’s September 2026 review of public COAs from 25 labs and one rating site’s certificate wrapper, 21 of 26 printed mg per vial. Only SideChain stated clearly that its mg excludes counter-ion and water. Counter-ions add weight in the same way as water; see TFA in peptides.

How Karl Fischer titration measures the water content of peptides

Karl Fischer published the method in 1935. Water reacts with iodine and sulfur dioxide in an alcohol, with a base to take up the acid formed. In methanol or ethanol, one molecule of water uses one molecule of iodine. A double platinum electrode detects the endpoint, and the titrator converts the iodine used into a mass of water. The method finds both surface water and water held inside the solid. A COA may cite USP general chapter <921> Water Determination, the US pharmacopoeia chapter for this test.

Karl Fischer vs loss on drying

KF is specific to water when no side reactions occur. Loss on drying weighs a sample before and after heating. Metrohm’s monograph notes that drying counts other volatile components and decomposition products as well as water, and needs a fixed temperature and time to give comparable numbers. A peptide powder can also hold residual solvents from synthesis and purification, which a drying result would count with the water. KF measures the water, and a separate residual solvents test measures the solvents.

Coulometric vs volumetric Karl Fischer

Both use the same reaction. They differ in how the iodine gets into the cell.

	Volumetric KF	Coulometric KF
Iodine source	Titrant from a burette	Made by an electric current from iodide in the cell
Calibration	Titrant strength (titer) must be determined again and again	Absolute method with no titer; checked with certified water standards
Range Metrohm gives	0.1% water and above	0.001% to 1%; ideal amount 10 µg to 10 mg of water
Sample handling	Solids can go straight into the vessel	Cell is never opened; solids are dissolved first or heated in an oven
Fit for one research vial	Suits larger absolute amounts of water	Suits the µg amounts in one vial

Metrohm’s monograph frames the choice in absolute terms: coulometry for small amounts of water, volumetry for larger ones. For a single vial, coulometry fits the scale. A cake of 2 to 10 mg at 1 to 3% water holds about 20 to 300 µg of water, inside Metrohm’s ideal coulometric range. The USP authors used coulometric KF for their lyophilised peptide lots. They opened the vials in a dry nitrogen atmosphere to keep moisture from the air out. Volumetric KF suits larger samples, such as portions of bulk powder.

The oven method for freeze-dried samples

Some samples dissolve poorly or react with KF reagents. For these, the sample is heated in an oven, often sealed in a vial, and a stream of dry gas carries the released water into the titration cell. Only the water reaches the reagent, so side reactions and matrix effects are ruled out. Sealed vials with PTFE-coated septa also keep air moisture out.

Metrohm’s monograph lists lyophilisates among the samples it analyses this way, at oven temperatures between 70 and 140 °C. The temperature must release the water without decomposing the sample. The oven can feed either a coulometric or a volumetric titrator.

What a good water content result shows

A water result you can check states:

1. The technique: coulometric or volumetric, direct or oven, and the oven temperature.
2. How the vial was opened: under dry gas or in room air.
3. The sample mass and the water found in µg or mg, as well as the %.
4. A standard check with a certified water standard on the day of the run.
5. Replicates, when there was enough powder for more than one.
6. The basis of the content result: “as is” or anhydrous, and whether water was measured at the same time.

KF is destructive. The powder used for water cannot be used for another test, so a full panel may need an extra vial. Our guide on how many vials to send covers this.

Which labs list a water content test

Labs call this test water content or moisture, and many peptide labs do not list it. These are labs from our September 2026 research, as their own sites describe them.

Lab	Water test on its site
ACS Peptide Testing Labs	Listed: “Karl Fischer moisture”, by coulometric or volumetric KF
TrustPointe Analytics	Listed: Karl Fischer water analysis, reported as % water
MDx BioAnalytical Laboratory	Listed: water content by Karl Fischer and by loss on drying
Ethos Analytics	Listed: solubility, pH and moisture testing; method not stated
Krause Analytical	Not listed on its peptide page
MZ Biolabs	Not listed
PharmLabs	Not listed for peptides

Not listing water is not a gap for most buyers: identity, purity and content per vial answer the common questions. Water matters when you need to account for the whole weight of the powder.

When a water test is worth adding

• A label that may mean powder weight. If content per vial reads below the label, water and counter-ion results show where the rest of the powder’s weight went.
• Powder bought by weight. Bulk powder sold by the gram includes its water. A water result tells you how much of what you weighed is water.
• Storage questions. A water result next to purity gives a baseline for later checks. See peptide degradation.
• In-house reference material. A powder used as a standard needs its water content before its content can be assigned.

Skip it when you only need to know which peptide is in the vial and how many mg it holds.

How HK Analytical tests water content

Our water content test uses Karl Fischer titration and reports water as % of the powder. You can add it to any sample alongside content per vial, counter-ion (TFA, acetate) and purity by HPLC, so one report shows the main parts of the powder’s weight. Results within days of receipt. Every number comes with its raw data, in a signed record you can check on our verify page. The example report shows the layout.