Direct spectrofluorimetric methods as alternatives to compendial ones used for the quality control of biopharmaceuticals: development, validation and application

Abstract A simple, rapid, precise, accurate and sustainable spectrofluorimetric method (SFM) was developed, validated and applied for the determination of 4-aminobenzoic acid and aromatic amino acids (phenylalanine, tryptophan and tyrosine). These compounds are used in biopharmaceutical formulations and therefore must be analyzed by quality control laboratories to meet the criteria established in pharmacopoeias. In general, potentiometric titration (PT) is described in the compendia as the official analytical technique. However, this method showed low sensitivity and selectivity, and moreover was performed with a non-aqueous solvent (acetic acid), which led to higher consumption of reagents and consequently to the formation of residues. Therefore, the SFM was developed in aqueous medium at pH 7.2 using phosphate buffer. It was successfully validated according to the ICH guidelines and showed good linearity range (r>0.999), specificity, accuracy and precision (within and between days) and robustness. The test results were compared between the SFM and PT using raw material samples, while according to the F- and t-tests at 95% confidence level, no statistical difference was found between the methods.

Enantiomeric Separation of Three Kinds of Aromatic Amino Acids by C18 Column Tandem Crown Ether Chiral Column / 分析化学

A reversed phase high performance liquid chromatographic ( RP-HPLC ) method was proposed by using C18 column tandem crown ether chiral column for simultaneous separation of three kinds of aromatic amino acid enantiomers. The chromatographic conditions, including mobile phase proportion, pH, column temperature and flow rate, were optimized. The experimental results showed that the three aromatic amino acid enantiomers could be successfully separated under the optimal conditions such as perchloric acid solution-acetonitrile ( 86:14, V/V, pH = 2 ) as mobile phase, column temperature of 20℃ and flow rate of 0. 4 mL/min. The separation under four HPLC column connection modes was further investigated. The results revealed that different kinds of amino acids could be separated on C18 column, but not for amino acid enantiomers; amino acid enantiomers could be separated on crown ether chiral column, but the chromatographic peaks of each amino acid enantiomers overlapped seriously; baseline separation of three amino acid enantiomers could be obtained under the connection of two columns, but the separation under different column connection sequence almost had no difference except the peak shape.

L-Phenylalanine and L-Tyrosine Catabolism by Thermophilic Geobacillus stearothermophilus.

Aims: This study investigated the potential of soil thermophile Geobacillus stearothermophilus for the biotransformation of phenylalanine and tyrosine. Study Design: G. Stearothermopilus grows well at 65ºC and has a good potential for transformation and biodegradation of many compounds including steroids, bile acids, tryptophan and other compounds. In this study G. stearothermophilus was harvested at mid-log phase at 65ºC, on tryptone yeast extract (TYE) medium. Cells were collected by centrifugation under aseptic conditions, washed with sterile water and suspended in phosphate buffer with phenylalanine or tyrosine as sole source of carbon at 65ºC. Metabolic parameters were optimized for optimal growth of the organism utilizing aromatic amino acids as an exclusive source of carbon. Methodology: The amino acid metabolites were exhaustively extracted with methanol from freeze dried broth. The concentrated pooled extracts were analyzed by thin layer chromatography (TLC) using polar solvent systems and purification of the extracts was achieved on preparative tlc plates and GC separations. The molecular structures of purified metabolites were established through spectral data. Results: Sixteen metabolites of phenylalainine and seventeen metabolites of tyrosine were identified in this study. Tyrosine metabolism extensively produced melanin pigments that caused hitches in the purification of tyrosine metabolites. Tyr metabolites were analyzed in cells cultured for short time. Conclusion: Our data suggest that G. stearothermophilus has a good potential to metabolize aromatic amino acids yielding hydroxylated, deaminated, decarboxylated and many other products. Oxidative metabolism of phenylalanine and tyrosine by a thermophilic G. stearothermophilus is being reported for the first time.