Loss on drying (revision 26)

Loss on drying determines the mass a sample loses when heated under defined conditions of temperature, pressure and time. It is simple, requires no specialised reagent, and is widely reported on peptide certificates.^[1]

It is not a water determination. The mass lost comprises water and any other volatile component — residual acetonitrile, acetic acid, ammonia from a salt-exchange step — so a loss-on-drying figure is an upper bound on water content rather than a measurement of it. Karl Fischer titration measures water specifically and will generally give a lower number on the same material.^[2]

For peptides the two are frequently confused, and a certificate reporting "water: 6.2% (LOD)" is reporting something other than water. The distinction matters because water is subtracted in calculating peptide content, and using a loss-on-drying figure for that subtraction understates the peptide.^[3]

A weighed sample is dried to constant mass under stated conditions — an oven at a specified temperature, or a desiccator over a specified desiccant, or under vacuum — and the mass difference expressed as a percentage of the starting mass.^[1]

The conditions are part of the result. Drying at 105 °C removes more than drying at 60 °C; drying under vacuum removes more than at atmospheric pressure. A figure quoted without its conditions is not comparable with another figure quoted without its conditions.

For peptides the conditions must be gentle enough to avoid decomposition. Aggressive drying can cause loss of mass through degradation rather than through evaporation, which inflates the figure and damages the sample; vacuum drying at moderate temperature is the usual compromise.^[3]

Peptide content is calculated by subtracting the non-peptide fractions — water, counterion, residual solvent, inorganic salt — from unity. Each must be determined by a method specific to it, or the subtraction double-counts or under-counts.^[3]

A certificate reporting loss on drying and residual solvents separately is at risk of double counting if both are subtracted, since the solvent contributed to the drying loss. A certificate reporting Karl Fischer water and residual solvents separately is not. This is the practical reason the compendial guidance on peptide quality attributes prefers the specific determinations.^[2]

Ordinary loss-on-drying figures for lyophilised peptides run from a few per cent to above ten. A high figure is not evidence of a defect: lyophilised peptide cakes are hygroscopic, and a material that has been opened, weighed and resealed will read higher than one analysed immediately.^[3]

A very low figure on a hygroscopic peptide is worth as much attention as a high one, since it implies either aggressive drying or a handling regime a purchaser cannot reproduce. Once a vial is opened in ambient air, water content begins to rise regardless of what the certificate said.

The practical consequence for anyone reconstituting material is that the certificate's water figure describes the lot as tested, not the vial as received, and a stability statement is written against a storage condition rather than against a vial's history. See Reconstitution of lyophilised peptides and Beyond-use date.^[1][4]

• Karl Fischer titration
• Peptide content
• Residual solvent
• Trifluoroacetate counterion
• Certificate of analysis