WATER ACTIVITY VS. KARL FISCHER TITRATION IN PHARMACEUTICALS: MEASURING MOISTURE THAT MATTERS

Your Karl Fischer result is within specification, yet the product still shows moisture-related degradation during stability testing. Two batches contain almost the same amount of water but behave differently over time, or a change in excipient improves stability even though the measured moisture content hardly changes. In other cases, a drying process continues to remove water without a clear indication of whether the additional drying is actually making the product more stable.

These are familiar situations in pharmaceutical development and quality control. Karl Fischer titration is one of the most established methods for pharmaceutical moisture analysis and remains highly effective when the question is how much water is present in a sample. The difficulty arises when total water content does not fully explain the behaviour of the formulation.

Water activity provides a different piece of information. Instead of measuring the quantity of water, it describes how thermodynamically available that water is within the product. This can be particularly relevant when the real concern is hydrolytic degradation, microbial susceptibility, caking, crystallisation, moisture migration, physical stability or the performance of the formulation during storage.

For pharmaceutical scientists, the distinction is therefore not simply between two analytical methods. The more useful question is whether total water content or water activity provides the better indication of the product risk that needs to be understood and controlled.

Moisture content and water activity answer different questions

Karl Fischer titration determines water content, typically reported as a percentage or in ppm. Depending on the method, it can provide highly sensitive determination of water and is well established for raw materials, pharmaceutical products, drying endpoints and applications where a compendial or registered moisture specification must be met.

Water activity, expressed as aw between 0.00 and 1.00 at a defined temperature, describes the thermodynamic availability of water rather than its total quantity. Water that is strongly associated with an excipient, crystal structure or other component of the formulation does not necessarily have the same ability to participate in chemical, physicochemical or microbiological processes as more freely available water.

This distinction is formally recognised by the United States Pharmacopeia. USP <921> covers water determination, whereas USP <922> specifically addresses water activity and explains that part of the total water in a pharmaceutical matrix may be tightly bound while another fraction is available to participate in processes such as hydrolysis or microbial growth. USP <1112> further describes the application of water activity to nonsterile pharmaceutical products, including formulation optimisation, reducing degradation of APIs susceptible to chemical hydrolysis and supporting microbiological risk assessment. [1–3]

The two measurements therefore cannot be converted into each other using a universal equation. The relationship between moisture content and water activity depends on the formulation, the interactions between water and the product matrix, and temperature. Two samples containing the same amount of water can consequently have different water activities, while a formulation containing more total water may have a lower aw if that water is more strongly retained.

That difference becomes particularly useful when a Karl Fischer result is technically correct but does not explain the stability behaviour observed in the product.

When more water does not mean less stability

A study on aspirin tablets illustrates the point particularly well. Researchers investigated aspirin formulations containing maize starch, microcrystalline cellulose and calcium hydrogen phosphate dihydrate and compared both the inherent moisture content and water activity of the excipients.

Maize starch contained more total moisture but had a relatively low water activity. Tablets containing maize starch subsequently showed lower aspirin degradation than formulations containing microcrystalline cellulose or calcium hydrogen phosphate dihydrate. The calcium hydrogen phosphate dihydrate had comparatively low moisture content but higher water activity and was associated with greater aspirin degradation. [4]

The practical implication is important for preformulation work. Selecting an excipient simply because it contains less total water does not necessarily create the most favourable moisture environment for a moisture-sensitive API. An excipient that retains water more strongly may contain more water while making less of it thermodynamically available.

Water activity therefore provides an additional way of evaluating excipient–moisture interactions. Instead of asking only which formulation contains the least water, development teams can investigate which formulation creates the most stable moisture environment for the API.

The same principle is relevant beyond aspirin. When hydrolysis, physical instability or another moisture-dependent mechanism is the concern, the objective is not necessarily to remove as many water molecules as possible. The objective is to understand which moisture state correlates with the critical quality attribute of the product and to control the formulation accordingly.

Using water activity to understand pharmaceutical stability

This approach can be incorporated directly into formulation and stability development. Water activity can be measured alongside Karl Fischer moisture content while degradation products, assay, caking, crystallisation, physical changes or other relevant stability parameters are monitored.

If two formulations have comparable Karl Fischer values but show different stability behaviour, their water activities may help explain the difference. If both moisture content and aw change during a stability study, comparing the two against the actual degradation profile can show which parameter better reflects the product response.

This is also why water activity should not be treated as a generic specification copied from another product. Its value is greatest when a product-specific relationship can be established between aw and a meaningful failure mechanism.

For a moisture-sensitive API, that may be degradation. For a powder it may be caking or flow behaviour, while for another formulation it may be crystallisation, physical instability or microbial susceptibility. The relevant water activity range should ultimately be connected to the quality attribute that determines whether the product remains acceptable.

Water activity is also relevant to advanced pharmaceutical formulations

The same concept is increasingly being investigated in complex pharmaceutical systems. A 2025 study published in Molecular Pharmaceutics evaluated water activity together with glass-transition temperature as indicators of long-term stability in lyophilised monoclonal antibody formulations. The work considered both residual moisture and the molecular interactions between water and excipients and used these parameters to identify formulation windows associated with favourable long-term stability. [5]

This is particularly relevant for lyophilised products because residual moisture content alone does not necessarily describe the complete moisture environment within the cake. The study concluded that no universally optimal residual moisture level exists independently of the excipient system, since the interactions between water and formulation components also influence stability.

For pharmaceutical development, this reinforces a broader point. Achieving the lowest technically possible residual moisture is not automatically equivalent to achieving the most stable formulation. Understanding both the amount of water and how that water interacts with the formulation can provide a more complete basis for development decisions.

From formulation development to process control

Water activity can also become useful when a moisture-related relationship identified during development is transferred into manufacturing.

Research on moisture-sensitive OPALMON tablets found a relationship between tablet water activity and drug stability. This relationship was subsequently used to develop a rotary vacuum-drying process at production scale, with water activity serving as a defined process target. The study demonstrated production-scale drying to below aw 0.03 within 30 minutes while reporting negligible tablet damage such as breaking or chipping. [6]

This example shows how water activity can move from being an analytical characterisation parameter to becoming a meaningful process-control parameter. A conventional moisture measurement can show how much water remains after drying, whereas a validated aw target can potentially indicate whether the product has reached a moisture state associated with the required stability.

The same approach can be considered during granulation, drying, coating or other manufacturing steps in which water is being removed, introduced or redistributed. If aw is shown to correlate with the relevant product outcome, it can help answer a much more practical question than simply how much water has been removed: has the process reached the condition required for product stability?

Moisture migration is another area where total water can be misleading

Pharmaceutical formulations frequently combine materials with very different water-binding properties. APIs, excipients, tablet coatings, capsule shells and other components may contain different quantities of water, while the packaged product can also interact with the humidity of the headspace and packaging system.

Comparing the percentage of water in two components does not tell you in which direction moisture will move. The driving force for moisture migration is the difference in water activity, with water tending to move from a region of higher aw towards one of lower aw until the system approaches equilibrium.

An excipient can therefore contain more total water than another material and still have a lower tendency to release moisture into it. This can be highly relevant when selecting excipients for a moisture-sensitive API or when investigating interactions between different components of a dosage form.

For formulation scientists, measuring aw can therefore provide information that Karl Fischer alone cannot deliver. Total moisture remains valuable for quantifying the water present, while water activity helps describe the potential for that moisture to redistribute within the system.

Packaging studies can benefit from the same information

Packaging development presents a similar challenge. A moisture-sensitive product may remain stable only while the packaging maintains the formulation within an acceptable moisture environment over its intended shelf life.

Karl Fischer can quantify changes in total moisture during storage. Water activity adds another perspective by indicating whether those changes move the formulation towards a moisture state associated with degradation, physical instability or microbial susceptibility.

This can make stability studies more informative. Rather than simply comparing how much moisture enters products stored in different packaging systems, development teams can trend aw alongside moisture content, assay, degradation products and relevant physical characteristics.

The resulting data can help determine whether a particular blister, bottle, barrier material or desiccant strategy maintains the product within its validated stability range. In this context, water activity does not replace conventional packaging studies but provides another way of linking package performance to actual product behaviour.

Water activity can support microbiological risk assessment

Water activity is also directly relevant to microbial growth, which is one reason it is specifically addressed in USP <1112>. The chapter identifies applications including improving the antimicrobial effectiveness of preservative systems, reducing susceptibility to microbial contamination and supporting scientific decisions relating to microbiological testing of nonsterile pharmaceutical products. [3]

This does not mean that measuring aw replaces an appropriate microbiological control strategy. Water activity is one factor within a broader product and process assessment, and any use for release or reduced testing needs to be scientifically justified within the applicable regulatory framework.

Its value is that it provides information directly related to the availability of water for microbial proliferation. Total water content alone cannot provide the same information because some of the water measured may be unavailable to microorganisms.

Where Karl Fischer and water activity add value across QA and QC

The value of the two methods becomes clearer when they are considered across the pharmaceutical lifecycle rather than as competing laboratory techniques. During incoming raw-material control, Karl Fischer is particularly useful for confirming total moisture against supplier or compendial limits, while water activity can provide additional insight into the functional moisture state of the material, its hygroscopic behaviour and its potential to exchange moisture during storage or handling.

During formulation development, Karl Fischer remains useful for following total water balance and understanding the moisture sensitivity of APIs and excipients. Water activity adds information about the interaction between moisture and formulation components and can help connect excipient choice, packaging and processing conditions with chemical, physical or microbial stability.

The distinction can also be useful during manufacturing. Karl Fischer can follow moisture removal during granulation, drying or coating, whereas water activity may help explain why materials with similar final water contents behave differently or whether a process has reached a moisture state previously linked to product stability.

For the finished product, total water content may remain the approved release parameter and should continue to be used wherever required. Water activity can nevertheless provide a complementary stability indicator for trending, packaging verification or investigations into moisture-related deviations.

The practical laboratory workflow can also be very different

For many QA and QC laboratories, the interest in water activity is not driven only by formulation science. Karl Fischer is a powerful and mature analytical technique, but the routine workflow can involve significant active analyst input depending on the sample and method.

A typical Karl Fischer workflow can include cell conditioning, reagent and solvent handling, drift management, careful sample transfer, cleaning and troubleshooting. Difficult or poorly soluble matrices may require extraction or KF oven methods, and chemical interferences or side reactions can require additional method optimisation.

The routine operating burden is also broader than the titrant itself. Depending on the laboratory, consumables can include syringes, needles, septa, vials, molecular sieves and other accessories, while solvents and chemical reagents introduce storage, PPE, ventilation and waste-management requirements. Maintenance can involve electrodes, seals, tubing, dosing hardware, pumps or generator components in addition to routine drift control. These are all relevant when comparing the real operating effort of moisture-analysis methods rather than looking only at instrument purchase price.

Water activity measurement has a different workflow. For many pharmaceutical samples, a representative portion can be placed directly into a sample cup and introduced into the measurement chamber without titration reagents, extraction or chemical sample preparation. The measurement then proceeds as the sample equilibrates at a controlled temperature, which means that much of the elapsed measurement time requires little active analyst intervention.

Routine consumables are typically limited to sample cups, certified humidity standards used for verification and calibration, and application-specific accessories such as filters where required. The measurement produces minimal chemical waste and avoids the routine handling of titration chemistry.

This does not mean that water activity measurement is free of analytical considerations. Temperature, equilibration, representative sampling and the influence of volatile compounds all need to be controlled, and the appropriate method must be validated for the intended application. A shorter hands-on workflow should also not be confused with an inherently shorter elapsed measurement time, since equilibration depends on the sample matrix.

The practical advantage is therefore more specific: for many routine applications, water activity can reduce active analyst handling while providing information that is directly related to moisture-driven product behaviour.

Comparing operating cost requires looking beyond reagents

The same principle applies when comparing cost per test. A meaningful comparison should include consumables, analyst time, waste handling, cleaning and maintenance rather than considering only the cost of a single reagent or sample cup.

Karl Fischer carries recurring costs associated with titrants, solvents or electrolytes, water standards and, depending on the method, syringes, septa, vials, molecular sieve and oven-related supplies. Difficult matrices and hazardous-waste requirements can further increase operating cost.

Water activity avoids routine titration chemistry, although it still requires sample cups, certified standards and occasionally application-specific accessories. Verification, calibration and normal instrument maintenance also remain part of the method.

The precise cost difference is highly site-specific because analyst rates, purchasing agreements, sample matrices, waste-management requirements and testing volumes vary considerably. For that reason, a laboratory considering water activity should calculate the complete local workflow rather than relying on a universal cost-per-test figure. The planning model developed for the accompanying QA/QC brief follows exactly this approach and treats the cost ranges as illustrative rather than as vendor quotations.

Water activity should complement Karl Fischer before it replaces it

The practical question for an established pharmaceutical laboratory is therefore usually not whether Karl Fischer should immediately be removed. Where a pharmacopoeial monograph, registered specification or validated analytical procedure requires total water content, Karl Fischer remains the appropriate method and the regulatory requirement continues to apply.

A more useful way to introduce water activity is to start with applications where existing moisture data leave an unanswered question. A development team may be trying to explain why formulations with similar Karl Fischer values show different degradation rates, why one excipient performs better than another, whether further drying actually improves stability, or why moisture uptake during a stability study affects one product more strongly than another.

In these situations, aw can initially be measured alongside the existing Karl Fischer result. The two measurements can then be trended against the relevant product outcome, whether that is chemical degradation, physical stability, microbial risk, moisture migration or packaging performance.

This creates a product-specific knowledge base rather than introducing an arbitrary new specification. If water activity shows a clear relationship with the critical quality attribute, the method can then be validated for its intended use, measurement conditions can be defined and meaningful product-specific limits can be established.

Once sufficient evidence exists, water activity may be expanded into routine QC or, in selected applications where scientifically and regulatorily justified, considered as an alternative to a conventional moisture-content test. This stepwise approach is also the most practical way to introduce aw without disrupting an established Karl Fischer control strategy.

Start with the problem your moisture result does not explain

For most laboratories, the strongest reason to investigate water activity is not that it represents a newer or simpler technique. It is that the existing moisture-content result sometimes does not answer the underlying product question.

If two batches have similar Karl Fischer results but different stability profiles, comparing their water activities may provide additional information. If an excipient with higher moisture content unexpectedly improves API stability, aw can help explain how that water is being held. If packaging studies show gradual moisture uptake, following aw can show whether that uptake is moving the formulation towards a region associated with product instability.

The same principle applies to drying and process development. If additional drying removes measurable moisture but no longer improves the parameter linked to product stability, continuing to dry may offer little benefit. Conversely, if aw remains above a validated stability range, a moisture-content result that appears acceptable may not tell the complete story.

Used in this way, water activity is not simply another analytical number. It becomes a practical tool for answering moisture-related questions that total water content cannot always resolve.

From measuring water to understanding moisture risk

Karl Fischer titration remains an essential technique in pharmaceutical moisture analysis. When the question is how much water a sample contains, particularly where a pharmacopoeial or registered specification requires total moisture, it provides exactly the information the laboratory needs.

Water activity answers a different question by describing how available that water is within the formulation. This can provide additional insight into hydrolytic degradation, microbial susceptibility, physical stability, moisture migration, packaging performance and the interaction between excipients and moisture.

The two methods should therefore not automatically be viewed as competitors. In many development and QA/QC applications, their greatest value comes from using them together until the relationship between moisture and the relevant critical quality attribute is understood.

The practical distinction is straightforward: Karl Fischer quantifies the water present, while water activity helps determine whether that water is in a state that matters to the product. When moisture content alone does not explain pharmaceutical stability or performance, that additional information can provide the missing link between the analytical result and the product risk that needs to be controlled.

Scientific references

Sekiya, N. et al. (2007). “Improved Stability of OPALMON Tablets under Humid Conditions. III: Application of the Rotary Vacuum Drying Method to Dry Opalmon Tablets.” Chemical & Pharmaceutical Bulletin, 55(4), 546–550. DOI: 10.1248/cpb.55.546. The authors reported a relationship between tablet water activity and drug stability and applied an aw target to a production-scale rotary vacuum-drying process.

United States Pharmacopeia, General Chapter <921>, Water Determination. This chapter covers pharmacopoeial approaches to determining water content and the use of water determination to demonstrate compliance with applicable monographs.

United States Pharmacopeia, General Chapter <922>, Water Activity. The chapter distinguishes total water from the water available to participate in chemical, physicochemical and microbiological processes and describes water activity measurement for pharmaceutical raw materials and products.

United States Pharmacopeia, General Chapter <1112>, Application of Water Activity Determination to Nonsterile Pharmaceutical Products. Applications include formulation optimisation, support for reducing hydrolytic degradation, microbial-risk assessment and scientific justification relating to microbial testing.

Veronica, N., Liew, C. V. & Heng, P. W. S. (2020). “Insights on the role of excipients and tablet matrix porosity on aspirin stability.” International Journal of Pharmaceutics, 580, 119218. DOI: 10.1016/j.ijpharm.2020.119218. The study compared moisture content and water activity of different excipients and found that higher moisture content did not necessarily correspond to greater aspirin degradation.

Zäh, M. et al. (2025). “Water Activity as an Indicator for Antibody Storage Stability in Lyophilized Formulations.” Molecular Pharmaceutics, 22(2), 918–926. DOI: 10.1021/acs.molpharmaceut.4c01106. The research evaluated water activity together with glass-transition temperature as indicators of long-term stability in lyophilised monoclonal antibody formulations.