The objective of this project is to develop methodology and validation data for some constituents of various injectable biological products including: (1) phenol used as a preservative in products such as allergenic extracts and Tuberculin PPD, (2) glycerin used in allergenic extracts as a preservative and/or stabilizer, (3) pyridine used in the manufacture of certain allergenic extracts, (4) m-cresol used as an anti-microbial preservative in bacterial vaccines, (5) 2-phenoxyethanol used as a preservative in inactivated Polio Virus Vaccine, (6) formaldehyde used as an inactivating agent in Influenza Virus Vaccine, Hepatitis B Vaccine, etc. and (7) chloride ion in albumin. A gas chromatographic method, incorporating an internal standard has been developed for phenol in Tuberculin PPD, Cholera Vaccine and allergenic extracts. An HPLC precolumn derivatization procedure has been developed for the determination of residual formaldehyde in viral vaccines and formaldehyde-modified allergenic extracts. Samples are directly derivatized with p-nitro benzylhydroxylamine (PNBA). The resulting derivative is separated on a reverse phase column and detected by UV at 254 nm. Coefficients of variation ranged from 1.5% to 6.3%. Recovery of added formaldehyde was 99.8% for an Influenza Virus Vaccine and 98.5% for a purified Tetanus Toxoid, Fluid product. The Schiff Base (Rosaniline HC1) colorimetric procedure yielded results that were in agreement with this method. An HPLC method for histamine has been developed that involves precolumn derivatization with o-phthaldialdehyde, reverse phase separation and fluorescence detection. This OPA-derivatization method has allowed the determination of low ppm levels of histamine found in the allergenic product, Histamine, Positive Skin Test Control.

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Intramural Research (Z01)
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