Faustinus K. Yeboah

Determination of toxaphene in biological samples using high resolution GC coupled with ion trap MS/MS

Abstract Toxaphene remains as a pollutant of global concern because of its persistence in the environment. Its complex combination of congeners and the lack of individual standards makes accurate measurement of toxaphene residues in environmental samples a challenge. We have developed a novel method to measure toxaphene in environmental samples using ion trap MS/MS. Adequate sensitivity for analysis of biological samples such as fish and marine mammal blubbers were achieved (around 0.02 ppb to 0.06 ppb for individual congeners and 5.6 ppb for toxaphene mixtures). Accuracy was demonstrated by good recoveries of spiked toxaphene in corn oil samples (102% for toxaphene congeners at 4 ppb and 96% for toxaphene mixture at 139 ppb level). Results of analysis of toxaphene in cod liver oil (NIST SRM 1588) using the MS/MS method compared favorably with the median round robin interlaboratory data. The major advantage of the ion trap MS/MS over the existing methods; electron capture negative ionization mass spectrometer or electron capture detector, is its excellent selectivity. Interference from other organochlorines can virtually be eliminated. Therefore, the developed MS/MS method can be used as a reliable alternative to high resolution mass spectrometer for measurement of toxaphene in environmental samples with relatively low cost instrumentation.

Lipase-catalyzed esterification of selected phenolic acids with linolenyl alcohols in organic solvent media

Lipase-catalyzed esterification of selected phenolic acids with linolenyl alcohols was investigated in selected organic solvent media. The enzyme activity for the esterification of dihydrocaffeic acid with linolenyl alcohol in solvent mixtures of hexane/2-butanone of 75∶25 (v/v) and 65∶35 (v/v) was 0.88 and 0.47 μmol of esterified dihydrocaffeic acid/(g of solid enzyme·min), respectively, with a corresponding esterification yield of 76 and 58%, respectively. However, the esterification of ferulic acid with linolenyl alcohol in the reaction medium of hexane/2-butanone of 65∶35 (v/v) resulted in a low yield (16%). Using the reaction medium of hexane/2-butanone of 75∶25 (v/v), an increase in linolenyl alcohol concentration with a concomitant use of a constant amount of dihydrocaffeic acid resulted in an increase in esterification yield. The highest esterification yield of 99% was obtained with a ratio of dihydrocaffeic acid to linolenyl alcohol of 1∶8 after 7 d of reaction. Biosynthesis of the end product, linolenyl dihydrocaffeate, was confirmed by electrospray ionization mass spectroscopy structural analysis; the esterproduct demonstrated an antiradical activity close to that of α-tocopherol.

Total toxaphene and specific congeners in fish from the Yukon, Canada.

Toxaphene is one of the major persistent organic pollutants with global environmental impacts. We have measured total toxaphene and specific congeners concentrations in 19 fish samples collected from the Yukon, Canada using gas chromatography coupled to ion trap MS/MS. The total toxaphene concentrations ranged from 42 to 242 ng/g (mean = 107+/-61 ng/g). The sum of the three specific congeners (Parlar 26, 50 and 62) was within 10-55 ng/g. The ratio of the sum of the three congeners to the total toxaphene varied between 8% and 25% in the fish samples but the ratio may be species specific. Our results suggest that consumption of these Yukon fish should have minimal risk of toxaphene exposure.

Efficient purification of paclitaxel from yews using high-performance displacement chromatography technique.

Paclitaxel was purified using high-performance displacement chromatography (HPDC) technique, but not by the mechanism of HPDC. On small scale, paclitaxel was extracted with methanol from dry needles of Taxus canadensis and was enriched by extracting with chloroform after removing water-soluble hydrophilic components and hexane-soluble hydrophobic components. Then, 93-99% purity of paclitaxel was obtained using the HPDC technique. On large scale, taxanes were enriched by solvent partitioning between acetic acid/MeOH/H(2)O and hexane and extracted with CH(2)Cl(2). Taxanes except paclitaxel were further removed by extracting with methanol-water-trifluoroacetic acid (1.0:98.9:0.1, v/v/v). Applying HPDC technique to water-insoluble substances is problematic as this method requires a highly aqueous solvent system. In order to overcome this incompatibility, a system was set up where paclitaxel, although in low concentration, was extracted by methanol-water-trifluoroacetic acid (10.0:89.9:0.1, v/v/v). Recycling the extracting solvent to ensure minimal volume, the extracted paclitaxel was adsorbed on a C(18) trap column. A C(18) column of 4.6mm internal diameter was then connected to the trap column. The HPDC technique was thus carried out using an isocratic acetonitrile-water-trifluoroacetic acid (30.0:69.9:0.1, v/v/v) mobile phase consisting of a displacer cetylpyridinium trifluoroacetate (3mg/mL). Paclitaxel was co-eluted with the displacer and spontaneously crystallized. The crystal (114mg) showed 99.4% purity and only 10% of paclitaxel in the starting crude extract was lost during the enrichment/purification processes. This large scale purification method was successfully applied to purify paclitaxel from Chinese yew in small scale, suggesting general applicability of the method. This is the first report of purifying a water-insoluble natural product using HPDC technique.

Tryptic fragments of phaseolin from protein isolates of Phaseolus beans

Crystalline and amorphous protein isolates from white kidney and navy beans (Phaseolus vulgaris) and baby lima and large lima beans (Phaseolus lunatus) were subjected to tryptic hydrolysis. The hydrolysis was monitored by the nitrogen solubility index (NSI) and reverse phase-high performance liquid chromatography/mass spectrometry (RP-HPLC/MS) analysis of the hydrolysates. The results of the NSI and RP-HPLC showed that the tryptic fragments of the crystalline isolates possessed higher solubility properties than the fragments of the amorphous isolates. The results of the MS study of phaseolin peptides in the RP-HPLC fractions of the hydrolysates showed that trypsin-specific bonds located in β-structured regions of β-phaseolin are resistant to tryptic hydrolysis, and that the most susceptible bonds to trypsin hydrolysis are located in regions not directly involved in the formation of secondary structural units, but in regions of disordered structure, or in regions interconnecting secondary structural units. Some trypsin-specific bonds located within α-helical structures were also cleaved by trypsin hydrolysis.

Synthesis of acylglycerols from ω-3 fatty acids and conjugated linoleic acid isomers

n−3 PUFA (ω−3 polyunsaturated fatty acid) concentrate from mackerel oil enriched in EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) was used to esterify isomers of CLA (conjuated linoleic acid) to produce acylglycerols (glycerides). Catalysis was potentiated by immobilized lipases from the yeast Candida antarctica and the mould Mucor miehei. C. antarctica lipase showed higher reactivity and much faster initial rate of incorporation of CLA into acylglycerols than its M. miehei counterpart. Synthesis with molecular sieves also achieved a better rate of incorporation of fatty acids into acylglycerols than using vacuum or systems without water removal. Esterification achieved at 40 °C with C. antarctica lipase was significantly different from that achieved at 60 or 50 °C. However, there was no significant (P<0.05) difference between esterification at 50 °C and 60 °C. The molar ratio of glycerol to fatty acid was found to influence the initial rate of incorporation, with the lower ratios showing higher initial rates compared with the higher ratios. The present study shows that direct esterification is an effective mechanism for producing acylglycerols from fatty acids in a controlled system.

Oxalic Acid-Induced Modifications of Postglycation Activity of Lysozyme and Its Glycoforms

The role of selected carboxylic acids and their potential to influence the glycation pattern and the enzymatic activity of lysozyme using glucose and ribose were investigated independently of the pH of the reaction medium. The model systems were incubated with and without selected carboxylic acids (maleic, acetic, oxalic, and citraconic) at 50 degrees C for 12 or 24 and 48 h at constant pH of 6.5. The effect of carboxylic acids on the glycation of lysozyme was studied by electrospray ionization mass spectrometry (ESI-MS) and by the measurement of the residual enzyme activity of lysozyme in the glycated samples. Of the carboxylic acids evaluated, oxalic acid showed the highest antiglycation activity. The residual lysozyme activity in both oxalic acid-glucose and oxalic acid-ribose systems was >80% compared with 46 and 36% activity in the controls of glucose and ribose systems, respectively. On the other hand, maleic, acetic, and citraconic acid containing systems with both sugars did not exhibit any enhanced enzyme activity relative to the controls. The results of this study show that oxalic acid was unique among the carboxylic acids evaluated with respect to its ability to interact with sugars and inhibit glycation.