Studies on factors influencing stability and recovery of paclitaxel from suspension media and cultures of Taxus cuspidata cv Densiformis by high-performance liquid chromatography.

An HPLC method was developed for quick scanning of taxanes from large numbers of plant cell suspension samples. The method was optimized for analysis of a range of taxanes of differing polarity. Identification of a standard mixture of paclitaxel and 12 related taxanes was achieved in less than 15 min using a gradient mode and a Microsorb-MV C8 column. The method was used to investigate the influence of several factors on stability and recovery of paclitaxel from suspension media and cultures of Taxus cuspidata cv Densiformis. Incubation time had the most significant influence on stability of paclitaxel, contributing 88% to the total variation. Shaking contributed 6% to the total variation. Light contributed only 0.25% to the total variation. Analysis of test samples of suspension cultures of T. cuspidata cv Densiformis over a 4 week period show paclitaxel, 10-deacetylbaccatin III, and baccatin III levels ranging from 0 to 149 microg/L, from 0 to 1.9 mg/L, and from 0 to 583 microg/L, respectively.

Negative fast atom bombardment ionization of aromatic sulfonic acids: unusual sample-matrix interaction.

The most intense ion(s) in negative ion fast atom bombardment (FAB) mass spectra of 2- and 4-benzaldehyde sulfonic acid (BSA) in glycerol or 3-nitrobenzyl alcohol matrix corresponds to a covalent association of the analyte with one or two matrix molecules accompanied by the elimination of a molecule of water. The molecular ion [M - H](-), however, is of low abundance. The identity of the resulting ions [M + nA - H(2)O - H](-) (where M is the analyte and A is the matrix) was confirmed by exact mass measurement using the peak matching technique. These covalent matrix-analyte complexes were not observed when the sulfonic acid functionality in BSA was substituted with COOH, NO(2), and OH or when the sulfonic acid was in salt form. These observations indicate that the free sulfonic acid group in BSA is responsible for the covalent adduct formation. To our knowledge, analyte-matrix covalent association in negative ion FAB spectra of BSA has not been reported previously.

Examination of recombinant truncated mature human fibroblast collagenase by mass spectrometry: identification of differences with the published sequence and determination of stable isotope incorporation.

Human fibroblast collagenase belongs to a family of matrix metalloproteinases which have been implicated in a number of connective tissue disorders ranging from rheumatoid arthritis to tumor invasion. To examine the active site of this enzyme by biophysical studies, a 19 kDa recombinant truncated mature collagenase (mCL-t) was prepared. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been utilized for the characterization of mCL-t. The molecular weights measured by these techniques identified the presence of two closely related protein components separated by approximately 100 Da. Edman sequence analysis demonstrated that the two protein components differ from each other by an amino terminal valine, consistent with the mass spectrometric data. In addition, the molecular weight of mCL-t determined by mass spectrometry did not agree with that calculated from the reported sequence. To identify the origin of this discrepancy, the DNA sequence of the mCL-t clone was examined. Several differences were noted between the DNA sequence of mCL-t and the published collagenase gene sequence. When these differences were taken into account, the measured molecular weights were found to be in good agreement with that calculated for the modified sequence. In separate experiments, both ESI and MALDI mass spectrometry have been used to determine molecular weights of mCL-t samples enriched with stable isotopes 15N and (15N + 13C). The measured molecular weights demonstrated a 97% (15N) and 99% (15N + 13C) incorporation of labeled isotopes in the two samples.