The goal of this project is to develop new or improved methods for the determination of moisture in biological products. A low residual moisture is necessary for the stability, viability and potency of the freeze-dried biological product. The residual moisture of freeze-dried biological products was first determined by the gravimetric or loss-on-drying method utilizing phosphorus pentoxide and vacuum at room temperature. This method has been optimized. For samples with uncomplicated thermogravimetric analysis (TG) curves, TG results have been shown to correlate with coulometric Karl Fischer results. Karl Fischer and TG moisture results may be different from the gravimetric moisture result for the same freeze-dried product due to the fact that different types of moisture (physically adsorbed or chemically bound moisture) are being measured. The thermogravimetric method has been used to determine the moisture content of Group A and Group C Meningococcal Polysaccharide bulks at levels of 5% to 25% moisture. Thermogravimetric/mass spectrometry (TG/MS) identified the TG transition corresponding to the loss of residual moisture in vaccines that have complex TG curves. Thermogravimetry provides precise heating conditions and weight loss information at specified temperatures while mass spectrometry identifies volatile compounds evolved during the weight loss process. A new TG/MS interface applicable to this analysis has been developed in our laboratory. The glass tubing interface connects the quartz combustion tube of the TG to the jet separator of the mass spectrometer. This interface allows continuous monitoring of the ion intensities of mass peaks m/e=18 (water) and m/e=44 (carbon dioxide) for the determination of residual moisture in freeze-dried biological products. Data has been collected clarifying thermograms for both Giant Short Ragweed Allergenic Extracts as well as Limulus Amebocyte Lysate Haemophilus b. Polysaccharide Conjugate Vaccines and other products such as Allergen Patch Test. A method is being researched that will determine moisture in space above freeze-dried cake in the vial. A new TG/MS capillary interface has been developed and applied to moisture analysis for AHF and BCG vaccine.